Receptor membranes

ABSTRACT

A membrane comprising a closely packed array of self-assembling amphiphilic molecules, and is characterized in that it incorporates a plurality of ion channels, and/or at least a proportion of the self-assembling molecules comprise a receptor molecule conjugated with a supporting entity. The ion channel is selected from the group consisting of peptides capable of forming helices and aggregates thereof, corronands, cryptands, podands and combinations thereof. In the amphiphilic molecules comprising a receptor molecule conjugated with a supporting entity, the receptor molecule has a receptor site and is selected from the group consisting of immunoglobulins, antibodies, antibody fragments, dyes enzymes and lectins. &#34;The supporting entity is selected from the group consisting of a lipid head group, a hydrocarbon chain(s), a cross-linkable molecule and a membrane protein. The supporting entity is attached to the receptor molecule at tan end remote from the receptor site. In preferred embodiments the ion channel is gramicidin A, and is preferable gated. Such membranes may be used in the formation of sensing devices.

This is a continuation-in-part of application Ser. No. 07/473,932 filed25 Jan. 1990, now U.S. Pat. No. 5,436,170, the disclosure of which isincorporated herein by cross-reference.

The present invention relates to membranes incorporating either receptormolecules and/or ion channels.

It is known that amphiphilic molecules may be caused to aggregate insolution to form two or three dimensional ordered arrays such asmonolayers, micelles, black liquid membranes, and vesicles or liposomes,which vesicles may have single compartment or may be of themultilamellar type having a plurality of compartments. It is also knownthat such amphiphilic molecules may be formed with cross-linkablemoieties. Under appropriate stimulus, such as UV radiation or ionisingradiation, the cross-linkable moieties can be caused to polymerise afterthe amphiphilic molecules have been caused to assume a suitably orderedtwo or three dimensional array. It is also known that suitable receptormolecules may be included in ordered arrays of amphiphilic molecules.

The selectivity and flux of ions through membranes can depend on thenumber, size and detailed chemistry of the pores or channels that theypossess. It is through these pores or channels that are permeatingsolute molecules pass across the membrane.

It is known that membranes may incorporate a class of molecules, callionophores, which facilitate the transport of ions across thesemembranes. Ion channels are a particular form of ionophore, which as theterm implies, are channels through which ions may pass throughmembranes.

Membranes incorporating ionophores exist in nature, and may also beproduced artificially. Australian Patent Application No 40123/85discloses the use of membranes including ionophores in biosensors. Theionophore exemplified in this reference is acetylcholine receptor. Theacetylcholine receptor functions as a gated ionophore in that itrequires the presence of acetylcholine before the acetylcholine receptoris able to facilitate the passage of ions across the membranes. Thissituation is similar to that encountered at nerve synapses.

The present invention consists in a membrane comprising a closely packedarray of self-assembling amphiphilic molecules, the membrane beingcharacterised in that (1) the membrane includes a plurality of ionchannels selected from the group consisting of peptides capable offorming helices and aggregates thereof, podands, coronands, cryptandsand combinations thereof; and/or (2) at least a proportion of theself-assembling amphiphilic molecules comprise a receptor moleculeconjugated with a supporting entity, the receptor molecule having areceptor site, the receptor molecule being selected from the groupconsisting of immunoglobulines, antibodies, antibody fragments, dyes,enzymes, and lectins; the supporting entity being selected from thegroup consisting of a lipid head group, a hydrocarbon chain(s), across-linkable molecule and a membrane protein; the supporting entitybeing conjugated with the receptor molecules at an end remote from thereceptor site.

The amphiphilic molecules are normally surfactant molecules having ahydrophilic "head" portion and one or more hydrophobic "tails".Surfactants may be any of the known types, i.e. cationic (e.g.quaternary ammonium salts), anionic (e.g. organosulfonate salts),zwitterionic (e.g. phosphatidyl cholines, phosphatidyl ethanolamines),membrane spanning lipid, or non-ionic (e.g. polyether materials). Theamphiphilic molecules are preferably such that they can be crossedlinked. For this purpose it is necessary to provide the molecules with across-linkable moiety such as a vinyl, methacrylate, diacetylene,isocyano or styrene group either in the head group or in the hydrophobictail. Such groups are preferably connected to the amphiphilic moleculethrough a spacer group such as is described in Fukuda et al, J. Amer.Chem Soc 1986 108, 2321-2327.

Polymerisation may be performed by any of the known methods forpolymerising unsaturated monomers, including heating with or without afree radial initiator, and irradiating with or without a free sensitiseror initiator.

In a preferred embodiment of the present invention the amphiphilicmolecules, not including a receptor molecule, include or are decoratedwith at least one moiety cross-linked with at least one correspondingmoiety on another of these molecules. In a further preferred embodimentthe ion channels and/or receptor molecule covalently linked to asupporting entity also include or are decorated with at least one moietycross-linked with at least one corresponding moiety on another molecule.

As stated above, the ion channels used in the present invention isselected from the group consisting of peptides capable of forminghelices and aggregates thereof, podands, coronands and cryptands.However, it is preferred that the ion channel is a peptide capable offorming a helix or aggregates thereof.

Synthetic ion channels have been described in the literature. Generallythese ion channels consist in covalently coupled dimers, trimers,tetramers or oligomers of podands, coronands or cryptands. Podands,cryptands and coronands have been described previously in the scientificliterature (see, for example, V F Kratgen et al, J Chem Soc Commun,1985, 1275; O E Sielcken et al, J Amer Chem Soc, 1987, 109 4261; J GNeevel et al, Tetrahedron Letters, 1984, 25 2263). The number ofoligomers are generally of a sufficient number that the synthetic ionchannel span a lipid bilayer membrane.

In the case of the single membrane spanning synthetic ion channels it isbelieved that gating of the ion channel conduction on addition of ananalyte may be achieved through the removal or addition of a weeklybound plugging or blocking moiety, that on binding of an analytemolecule onto a receptor molecule, said receptor molecule being boundonto the ion channel and having the plugging or blocking moiety attachedto the receptor, removes the plug or block thus allowing ions to flowthrough the channel. This change in ion flux may be readily monitoredand is related to the concentration of the analyte solution.

Alternatively, a receptor group may be coupled onto the synthetic ionchannel such that on binding of the receptor to the analyte aconformational change is induced in the ion channel such that the ionsflux through the channel is changed. This change in the ion flux canthen be monitored and is related to the concentration of the analyte insolution.

Synthetic ion channels of the membrane spanning type are disclosed by R.M. J. Nolte et al., Israel Journal of Chemistry, Vol. 24, pp. 297-301,1984; J. Chem. Soc., Chem Commun., pp. 1275-1276, 1985; TetrahedronLetts., Vol. 25, No. 21, pp. 2263-2266, 1984; J. Am. Chem. Soc., Vol.109, pp. 4261-4265, 1987; J-M. Lehn et al., Can. J. Chem. Vol. 66, pp.195-200, 1988; A. Nakano et al., J. Am. Chem. Soc., Vol. 112, pp.1287-1289, 1990; N. Voyer, J. Am. Chem. Soc., Vol. 113, pp. 1818-1821,1991; M. J. Pregel et al., Agnew. Chem. Int. Ed. Engl., Vol. 31, pp.1637-1640, 1992; V. E. Charmichael et al., J. Am. Chem. Soc., Vol. 111,pp. 767-769, 1989; T. M. Fyles et al. Tetrahedron Letts. Vol. 31, pp.1233-1236, 1990; Y. Kobuke et al., J. Am. Chem. Soc., Vol. 114, pp.7618-7622, 1992; Iimori et al J. Am. Chem. Soc. Vol. 111, 3439-3440,1989 and Sansom Prog. Biophys. Mol. Biol. Vol. 55, 139-255, 1991.

However, as for the case of gramicidin ion channels where two halfmembrane spanning ion channels are needed to complete a conductingmembrane spanning ion channel, the synthetic ion channels may besimilarly constructed such that two halves of the synthetic ion channelmay align in the membrane to form a conducting ion channel. Thus in thisform the synthetic the synthetic ion channels are in dynamic equilibriumbetween the unaligned monomeric, half membrane spanning, non-conductingstate and the aligned, membrane spanning, conducting state. Suchhalf-membrane spanning synthetic ion channels may have groupsincorporated into the end of the ion channel that is inserted into themembrane such that it is capable of forming hydrogen bond or othernon-covalent bond between the two synthetic ion channel halves.

Peptides which form a helice sgenerally need to exist as aggregates inthe membrane to form ion channels. Typically, the α helical peptidesarrange to form aggregates in such a manner that an ion channel iscreated through the aggregate.

It is preferred that the ion channels is peptide which forms a β helix.An example of such peptide is the polypeptide gramicidin A. The primarysequence of gramicidin A is shown FIG. 1. This molecule has been thesubject of extensive study (for further information see Cornell B A,Biomembranes and Bioenergetics (1987), pages 655-676). The ion channelgramicidin A functions as a polar channel which traverses non-polarbiological membranes. It is produced either synthetically or extractedfrom Bacillus brevis, in phospholipid bilayers gramicidin A is thoughtto exist as a helical dimer which substantially partitions into thehydrophobic region of the bilayer.

When it is desired to cross-link the amphiphilic molecules and thegramicidin A, gramicidin A may be modified by replacing one, two, threeor four tryptophan groups in the gramicidin A with polymerisable group,such as styrene. The polymerisable group is attached to the alpha carbonof the 9, 11, 13 and/or 15th amino acid residue of the native gramicidinA.

Furthe rexamples of molecules which may be used as ion channels in thepresent invention include gramicidin B, gramicidin C, gramicidin D,gramicidin GT, gramicidin GM, gramicidin GM⁺, gramicidin GN⁻, gramicidinA' (Dubos), band three protein, bacterionhodopein, mellitin,alamethicin, alamethicin analogues, porin, tyrocidine, and tryothricin.

Hereafter, the family of gramicidins will be referred to as simplygramicidin.

In the particular case of gramicidin, when the membrane is a monolayer,a monomer of gramicidin A could be used as the ion channel. In thesituation where the membrane is a bilayer, a synthetic analogue ofdimeric gramicidin A could be used the ion channel. This syntheticanalogue could be provided with suitable cross-linkable moieties. Inaddition, where the membrane is a bilayer the ion channel may consist oftwo gramicidin A monomers, in which each monomer is in a differentlayer. In this situation the gramicidin A monomers are able to diffusethrough the layers and when the two monomers come into alignment an ionchannel is formed through the bilayer.

While the membranes of the present invention incorporating ion channelsmay be used as membrane coatings having high conductance, in a number ofapplications it is necessary for the conductance at the membrane to beresponsive to the presence of an analyte. Therefore, in a preferredembodiment of the present invention, the ion channel is gated by areceptor moiety attached to, or associated with, an end of the ionchannel, the receptor moiety being such that it normally exists in afirst state, but when bound to an analyte exists in a second state, saidchange of state causing a change in the ability of ions to pass throughthe ion channel.

The first state of the receptor moiety will normally be a state in whichthe passage of ions through the ion channel is prevented or hindered.Attachment of the analyte to the receptor will this cause the receptorto enter the second state wherein ions may pass through the ion channel.In this arrangement an ion channel may be used to detect as little as asingle molecule of analyte. The attachment of a single molecule ofanalyte will cause an ion channel to open and thus cause a leak of ionsacross the membrane. After a brief time this ion leak may be detected asthe signal for the binding of the analyte to the receptor. Themeasurement of current flow across membranes due to a single ion channelis known and typically yields a current of 4 pA per channel.

As would readily be appreciated by a person skilled in the art thealternative arrangement is when the receptor moiety is in the firststate ions are able to pass through the ion channel and when in thesecond state the passage of ions through the ion channel is prevented orhindered.

The receptor moiety may be any chemical entity capable of binding to thedesired analyte and capable of changing the ion channel form its firststate to its second state upon binding to that analyte. The receptormoiety is any compound or composition capable of recognising anothermolecule. Natural receptors include antibodies, enzymes, lectimes, dyesand the like. For example the receptor for an antigen is an antibody,while the receptor for an antibody is either an anti-antibody or,preferably, the antigen recognised by that particular antibody.

In a preferred embodiment the receptor moiety is attached to the ionchannel, and preferably comprises a peptide end sequence on apolypeptide ion channel, which end sequence can bind to an antibody. Theantibody binding causes the shape of the end sequence to change thuspermitting ions to flow through the ion channel. The receptor moiety maybe attached to the ion channel. The receptor moiety may be attached tothe ion channel in any suitable way and is not necessarily formedintegrally therewith. The receptor may thus be covalently ornon-covalently conjugated with the ion channel.

The analyte may be any suitable molecule or group of molecules whichwill bind to the separator moiety and cause it to change its position,spatial configuration or change so as to convert the ion channel fromthe first to the second state. If the receptor is a peptide then theanalyte might be an antibody or immunoglobulin, an enzyme or cellsurface receptor. If, however, the receptor were the antibody or enzymethen the anlyte might be any molecule that binds thereto.

In the embodiment described above, the receptor moiety in its firststate, effectively occludes the ion channel. A variation on this methodof gating is to provide on the receptor moiety a compound capable of"plugging" the ion channel. In this embodiment binding the analyte tothe receptor moiety effectively pulls the plugging compound out the ionchannel, thereby enabling ions to pass through the channel. Suitableplugging compounds include the receptor itself or smaller molecules egethylammonium ions, and methylammonium ions which totally block the ionchannel, as do divalent ions, such as the calcium cation. In additionguanadinium ions are effective as partial blockers of ion channels,whilst it is suspected that compounds such a phenols, ethanolamines andlonger chain versions of ammonium ions may also be useful as pluggingcompounds.

In general, it is believed that positively charged species with an ionicdiameter of 4 to 6 Angstroms attached to a receptor moiety of sufficientflexibility to allow the ion to enter and "plug" the channel may beused.

In a further preferred embodiment the receptor moiety is an antibody, orpreferably an antibody fragment including at least one Fab fragment(hereinafter Fab). In this arrangement, in the first state, Fab allowsthe passage of ions through the channel, and in the second stateprevents the said passage of ions;

In a situation where the ion channel is dimeric gramicidin A and thereceptor is Fab attached to the ion channel, without wishing to be boundby scientific theory, it is believed that the passage of ions throughthe channel is blocked upon the binding of the Fab to the analyte (i.e.second state) due to disruption of the dimeric gramicidin A backbone, orto disruption of the portion of the helix of the dimeric gramicidinattached to the Fab.

In the present invention, where at least a proportion of the amphiphilicmolecules comprise a receptor molecule conjugated with a supportingentity, the receptor molecule and the entity together form a newamphiphile. This enables the formation of a membrane having a highdensity of receptor sites. In principle, the density of receptor sitesis limited solely by consideration of steric hindrance between theindividual receptor molecules.

In a preferred embodiment of the present invention when the receptormolecule is an antibody fragment, the antibody fragment includes atleast one Fab fragment. In a further preferred embodiment of the presentinvention the supporting entity is either a cross-linkable molecule ormembrane proteins, and preferably the cross-linkable molecules isbi-functional. In another preferred embodiment of the present inventionthe antibody fragment consists of two Fab fragment, each Fab recognisinga different antigen. In yet another preferred embodiment the antibodyfragment consists solely of the Fab fragment.

In a further preferred embodiment of the present invention where aproportion of the amphiphilic molecules comprise a receptor moleculeconjugate with a supporting entity, the membrane may include receptorseach reactive with a different molecule or antigenic determinant. Forexample where the receptor molecules are a mixture of two differentFabs, it is possible for half the receptors to be directed against oneantigenic determinant and the other half to a different antigenicdeterminant.

An immunoglobulin is a Y-shaped molecule composed of four polypeptidechains (two light and two heavy chains) linked by disulphide bonds. Thelight and heavy polypeptide chains are folded into globular regionscalled domains. The portion between the C.sub.γ1 and C.sub.γ2 domaine ofthe heavy chains, nown as the hinge region (Smyth, D. S and Utsumi, S.(1967) Nature 216, 332) can be cleaved by proteolytic enzymes. Cleavageby the enzyme papain releases the two arms of the Y-shaped molecule,i.e., the Fab fragments, from the remaining F_(c) stem portion(Zappacoeta, S et al (1968) J Immunol 100, 1268). The Fab fragments arepurified by ion exchange chrometography (Notkins, A. L. et al (1968) JImmunol. 100, 314) and affinity chromatography (De La Farge, F et al(1976) J. Immunol 123 347). In another preferred embodiment of thepresent invention the Fab fragment is linked to the entity selected fromthe group comprising lipid head group, a hydrocarbon chain(s),bi-functional cross-linker molecules, and membrane proteins, by means ofa covalent bond. Such a covalent linkage takes advantage of the variouslinkage sites available in the Fab proteins. Groups such as thesulfhydryl group of cysteine residues, α- amino, ε- amino groups oflysine residues, phenolic hydroxyl groups of tyrosine residues and theimidazole groups of histidine residues, are available for conjunctionwith any of the entities listed above. Once bounded to the lattermolecules, a new amphiphile is formed, and the Fab fragments are henceanchored in the membrane and act as highly sensitive and stabledetectors of any chosen antigen presented to the membrane surface.

A particularly preferred membrane protein is the polypeptide gramicidinA. This polypeptide commonly exists in membranes as a dimer.

As would be appreciated by persons skilled in the art recombinant DNAtechnology may be useful for the preparation of membraneprotein-receptor conjugates.

In yet another preferred embodiment of the present invention at least aproportion of the amphiphilic molecules consist of the receptormolecules convalently linked to a hydrocarbon(s). Preferably thereceptor molecule is an Fab fragment. By changing the number ofhydrocarbon chains it is possible to change the phase of the membrane.Some of the membrane phases which may be achievable by varying thenumber of hydrocarbon chains are the Hexagonal τ (H_(I)) phase, wherethe membrane forms tubes in which the interior is hydrophobic and theexterior polar, and Hexagonal ⊥⊥ (H.sub.ττ) phase where the membraneforms tubes in which the exterior is hydrophobic and the interiorhydrophilic. Other membrane structures which may be formed are lamellaeand micelles.

The use of hydrocarbon chains also lends itself to the binding of themembrane to solid surfaces such as ceramics, oxides, hydrogel, silicon,polymers, and transition metals, in the formation of a biosensor.Preferred transition metals include palladium, gold and platinum. Thismay be achieved by non-covalent attraction, or by covalent reactions.Thus, vinyl groups vinyl-terminated lipid, a sulphur-terminated lipidcould be adhered to a metal (e.g. gold or pelladium) substrate, ifnecessary, can be achieved using any of the known techniques such assilylation of silica surfaces.

As stated previously, the present invention provides a membrane having ahigh density of receptor sites. This is particularly true where thereceptor molecule is an Fab fragment. Using Fab fragments as thereceptor molecules it is possible to achieve a membrane in which up to2% of the amphiphilic molecules include a Fab fragment. Preferably, fromabout 1% to about 2% of the amphiphilic molecules include a Fabfragment.

The membranes according to the present invention in which at least aportion of the amphiphilic molecules comprise a receptor molecule boundto an entity, are particularly useful in biosensors. These membranesserve as highly selective binding surfaces to which molecular species tobe detected will bind. The binding of the molecule to be detected may beregistered by any one of a number of know techniques including:

(a) Ellipsometry,

(b) Fluoresence polarisation,

(c) Internal reflectometry,

(d) Light scattering,

(e) Surface plasmon resonance,

(f) Surface acoustic wave modification,

(g) Potentiometric effects i.e. changes in voltage,

(h) Amperometric effects i.e. changes current,

(i) Thermal effects i.e. a change in temperature or heat flow,

(j) Mass or density changes as may be reflected, for example, in thefrequency change of a piezolectric device,

(k) Measurement of membrane phase change,

(l) Radio immunoassay, and

(m) Enzyme-linked immunoassay.

In the situation where the membrane includes an ion channel and in whichat least a porportion of the self-assembling amphiphilic moleculescomprise receptor molecules conjugated with a supporting entity, it ispreferred that the ion channel is gramicidin A, and preferably thereceptor molecule is an antibody or antibody fragment including at leastone Fab fragment.

In a further preferred embodiment of the present invention the ionchannel is gated. In this embodiment the membrane includes an ionchannel bound to an analyte in close proximity to an amphiphilicmolecule comprising a receptor attached to an entity, the receptor beingcapable of binding to the analyte. In the absence of any free analyte,the analyte bound to the ion channel is attached to the receptor,thereby enabling ions to pass through the ion channel. Upon the additionof free analyte, there is competition for binding to the receptor, andthe analyte bound to the ion channel is released from the receptor,thereby blocking the passage of ions through the ion channel.

In an alternative embodiment the membrane includes an ion channel inclose proximity to an amphiphilic molecule comprising a receptormolecule conjugated with a supporting entity. When the receptor moleculeis not bound to an analyte it at least partially occludes the ionchannel thereby preventing or hindering the passage of ions through theion channel. Binding of the analyte to the receptor moiety causes achange in the receptor moiety which removes the occulusion of the ionchannel thereby allowing ions to pass therethrough. In this embodimentit is preferred that the membrane is a bilayer, the ion channel is twogramicidin monomers or a covalently linked dimer and the receptor moietyis a Fab fragment.

In a further preferred embodiment a linker group is provided between thereceptor molecule and the entity and/or between the ion channel andreceptor moiety. The linker group attached to the head group of thelipid should be of sufficient length to allow reaction of the receptormolecule by reducing steric hindrance that occurs close to the lipiditself. The linker is preferably not hydrophobic so that it can extendtoward the aqueous receptor molecule, and should be terminated with agroup amenable to attachment of the receptor molecule. For example, thethiol group of a Fab is suited to attachment to linker-bound melaminegroups or to electrophiles such as alkyl halid groups. Similarconsiderations arise in devising linker groups for attachment ofreceptor moieties to ionchannels such as gramicidin A.

The membranes of the present invention have particular application inthe production of biosensors. Accordingly, a preferred embodiment of thepresent invention provides a biosensor incorporating the membrane of thepresent invention.

Methods for measuring the change in conductance of self-assemblingmembranes, with and without ionophores, are comprehensively described inthe scientific literature, and include the use of black lipid membranechambers, electodes in which a monolayer, bilayer or bulk lipid is madeto coat porous papers, polymers or ceramics and patch clamping in whichapproximately 1 to 10 ion channels are incorporated in a monolayer orbilayer supported on a microelectrode. The method of signal analysis canbe a two, three or four terminal impedance measurement in which thefrequency characteristics, noise spectra, cyclic voltammetry orstatistics on the inherent making or breaking of ion channels are usedto characterise changes in conductance through the membrane.

One of the major difficulties encountered to date in the use ofmembranes in the formation of biosensors has been the fragility of themembrane. The present inventors have overcome this difficulty and formedthe membrane of the present invention on a solid surface and found thatthis membrane is robust.

This method involves providing on the membrane and the solid surfacegroups capable of reacting with each other. The preferred solid surfacesinclude hydrogel, ceramics, oxides, silicon, polymers and transitionmetals. Preferred transition metals are gold, platinum and palladium. Apreferred solid surface in the formation of a biosensor comprising amembrane bilayer attached to a solid surfacem the bilayer having anupper and lower layer, the lower layer being adjacent the solid surfaceand being provided with groups reactive with the solid surface or withgroups provided thereon; each layer of the bilayer being composed ofself-assembling amphiphilic molecules and gramicidin monomers, andwherein a proportion of the amphiphilic molecules in the upper layercomprise a receptor molecule conjugated with a supporting entity, thereceptor molecule having a receptor site, the receptor molecule beingselected from the group consisting of immunoglobulins, antibodies,antibody fragment, dyes, enzymes and lectins, and the supporting entitybeing selected from the group consisting of a lipid head group, ahydrocarbon chain(s), a cross-linkable molecule, and a membrane protein;the supporting entity being conjugated with the receptor molecule at anend remote from the receptor site.

In this embodiment it is preferred that the receptor molecule is anantibody fragment and preferably is a Fab fragment. It is also preferredthat the solid surface is a palladium-coated glass electrode.

In a further embodiment the present invention provides a biosensorcomprising a membrane bilayer attached to a solid surface, the bilayerhaving an upper and lower layer, the lower layer being adjacent to thesolid surface and being provided with groups reactive with the solidsurface or with groups provided thereon; each layer of the bilayer beingcomposed of self-assembling amphiphilic molecules and gramicidinmonomers; and wherein a receptor moiety is attached to the gramicidinmonomers in the upper layer.

As would be appreciated by a person skilled in the art, in a biosensor,reducing the area of the membrane will increase the sensitivity andselectivity of the sensor by improving its signal dynamic range and bandwidth.

The present invention will now be described by way of reference to thefollowing figures in which:

FIG. 1 shows the structure of gramicidin A;

FIG. 2 shows a schematic representation of a gated ion channel;

FIGS. 3 to 5 illustrate the manner in which various membrane phases areobtainable;

FIG. 6 is a graph showing the detection of an analyte using a membranebiosensor of the present invention formed as per Example 8.

As shown in FIGS. 4 to 5, 10 represents the receptor molecule and 11represents a hydrocarbon, non-polar moiety of variable length from 8carbons to 25 carbons containing methylene, alkene, alkyne orsubstituted aromatic compounds. X represents the number of hydrocarbongroups attached or associated with the receptor moiety 10. This numberwill depend on the bulk of the polar group. For antibody fragments Xtypically equals approximately 50 in order to achieve a planar membrane.The schematic geometry the dictates the long range structure formed bythe aggregated amphiphilies. As is shown in FIG. 3 when the number ofhydrocarbon groups is large a hexagonal phase II membrane is produced.FIG. 4 shows that when the number of hydrocarbon groups is approximately50 a lamellar phase membrane is produced whilst FIG. 5 shows that whenthe number of hydrocarbon groups is small a micellar phase membrane isproduced.

The present invention will now be described with reference to thefollowing example:

EXAMPLE 1 Synthesis of Cross-Linkable Moieties

(i) Synthesis of p-Hydroxystyrene

p-Hydroxyxstyrene was converted to 1-(p-acetoxyphenyl)ethanol and thendehydrated using liquid phase dehydration in the presence of potassiumacid sulfate to produce p-acetoxystyrene, according to the method ofCorson et al (J. Org. Chem. 1958 23 544). p-Acetoxystyrene (1.6 g) wasadded to a stirred solution of potassium hydroxide (1.4 g) in water (14ml) at 5 degrees Centigrade. Stirring was continued at 0-5 degreesCentigrade for 2 h. The mixture was then washed with ether, and theaqueous phase neutralised with saturated sodium hydrogen carbonatesolution. The product was extracted into dichloromethane, the solutionwas dried over anydrous calcium chloride and the solvent removed, toyield a cloudy oil (0.7 g) which solidified on standing to a waxy solid.

(ii) Synthesis of Methyl 11-(p-Vinylphenoxy)undecanoate

Hydrogen chloride gas was bubbled through a stirred solution of11-bromonudecanoic acid (2.65 g) in methanol (20 ml) for 1 h at roomtemperature. The solvent was then removed and the residue in ether waswashed with water, dried over anydrous sodium sulfate and the solventremoved. The residual pale oil (2.8 g, 100%) was identified as methyl11-bromonudecanoate.

This was converted to 11-(p-vinylphenoxy)undecanoic acid by the methodof Hasegawa et al (Polym. Bull, 1985, 14 31).

(iii) Synthesis of1-0-(11-(p-Vinylphenoxy)undecanoly)-2-0-octadecylglycerol

The method of Hasegawa et al (Polym. Bull., 1985 14, 31) was followed,however, the condensation step was allowed to react for 5 days, and theproduct was chromatographed on silica gel, eluting with ether/lightpetroleum (1:3). The total product from 0.92 g 11-(p-vinylphenoxy)undecanoic acid was 1.25 g (66%).

EXAMPLE 2 Synthesis of Linker Group for Attachment to Lipid or IonChannels

(i) 11-Chloro-3,6,9-trioxaundecon 1-ol

1,8-dichloro-3, 6-dioxaoctane was prepared from triethylene glycol,thionyl chloride and pyridine according to the method of C J Pedersen(J. Am Chem. Soc., 1967, 89 7017), b.p. 121°-122° C./15 mm Hg.

A solution of 1,8-dichloro-3, 6-dioxaoctane (40 g) and potassiumhydroxide (11.0 g) in ethylene glycol (100 ml) was stirred at 100° C.for 18 h. The mixture was then cooled, filtered and the residue washedwith acetone (2×35 ml). The combined filtrate was then distilled toyield the product as a clear oil (13.5 g, 30%), b.p. 121°-122° C./15 mmHg; I.r. (liquid film) 3430 cm⁻¹.

(ii) 11-Chloro-3,6,9-trixaudec-1-yl succinate

A solution of 11-chloro-3,6,9-trixaundecan-1-ol (2.00 g), succinicanhydride (0.941 g), pyridine (0.10 ml) and dimethylaminopyridine (0.02g) in tetrahydrofuran (10 ml) was refluxed for 24 h. The mixture wascooled and the solvent was removed to yield the product as a clear oil(2.9 g, 100%). I.r. (liquid film): 3000 (b, CO² H), 1730 (C═O) cm⁻¹.

EXAMPLE 3 Attachment of Linker Group to Lipid

(i) 1-0-(11-(p-Vinylphenoxy)undecanoyl)-2-0-octadecyl3-0-(11-chloro-3,6,9-trixaundec-1-yl succinatoyl)glycerol

11-Chloro-3,6,9-trixoaundec-1-yl succinate (0.60 g) was dissolved inthionyl chloride (5 ml) and refluxed for 3 h. Excese thionyl chloridewas removed, toluene (5 ml) was added and removed at 0.1 mm Hg to yieldthe carboxylic acid chloride as a pale yellow oil (0.64 g, 100%). I.r.(liquid film); 1785 (COCl), 1730 (C═O) cm⁻¹.

A solution of the carboxylic acid chloride (0.15 g) in tetrahydrofuran(0.5 ml) was added dropwise to a solution of1-0-(11-(p-vinylphenoxy)undecanoly)-2-0-octadecylglycerol (0.300 g) andpyridine (0.10 ml) in tetrahydrofuran (5 ml). The mixture was stirred atroom temperature for 18 h and then poured onto was (75 ml). The combinedchloroform extracts were washed with sulfuric acid (5%, 50 ml) and brine(50 ml), dried (MgSO₄) and evaporated. The crude product waschromatographed on silica, using ethyl acetate/light petroleum. 40:60v/v as eluent, to yield the product as a clear oil, which solidified onstanding (0.215 g, 49%). I.r. (liquid film) 1730 (C═O) cm⁻¹.

(ii) 1-0-11-(p-Vinylphenoxy)undecanoyl!-2-0-octadecyl-3-O-acetoylglycerol

A mixture of 1-0- 11-(p-Vinylphenoxy)undecanoyl!-2-0-octadecylglycerol(0.20 g), redistilled acetic anhydride (3 ml) and pyridine (0.2 ml) wasstirred at room temperature for 18 h. Excess acetic anhydride wasdistilled and the residue was taken up in chloroform (40 ml). Thechloroform was washed with sodium hydrogen carbonate solution (5%, 2×50ml), hydrochloric acid (5%, 50 ml) water (50 ml), dried (MgSO₄) andevaporated to yield the product as a colourless oil (0.16 g, 74%) whichwas homogeneous by t.l.c. IR 1735 cm⁻¹ (c═O).

Hereafter this compound is referred to acetate lipid.

EXAMPLE 4 Attachment of a Linker Group to Gramicidin A

A mixture of gramicidin (0.0633 g), 11-chloro-3,6,9-trioxaundec-1-ylsuccinate (0.032 g), dicyclohexyldiimide (0.021 g) anddimethylaminopyridine (0.020 g) in dichloromethane was stirred at roomtemperature for 24 h. The mixture was then washed with water (×50 ml),dried (MgSO4) and evaporated. The crude product was purified bypreparative layer chromatography using dioxane as eluent to yield thegramicidin analogue (hereafter gramicidin R) as a white solid 0.30 gI.R. 1725 (CO2) 1625 (CONH)^(cm-1).

EXAMPLE 5 Preparation, Isolation and Characterisation of Fab Fragments

IgG antibodies were purified from ascites fluid by chromatography onProtein A to a single band on SDS polyacrylamide gel electrophoresis.

Fab2 fragments were prepared from pure antibodies by pepsin digestion(1:100 enzyme: antibody weight ratio) for 30 minutes at pH 4.2cation-exchange chromatography yielded the pure active Fab2 fragments asestablished by the single band of 100,000 molecular weight mark on SDSpolyacrylamide gel electrophoresis. Electrophoresis under reducingconditions showed a band at 25,000 molecular weight corresponding to thelight chain and heavy chains of the two Fab' components of Fab2.

Fab' were obtained from Fab2 by modification of the method of Martin F Jet al (Biochemistry, 1981, 20, 4229-30). Fab2 were reduced with 20 mMdithiothreitol at ph 5.5 for 3 hours at room temperature. Dithiothreitolwas removed by ultrafiltration using membranes with 30,000 molecularweight cut-off range. Fab' possessed comparable antigen bindingactivities to Fab2 and gave a single band at the 50,000 and 25,000molecular weight markets when SDS electrophoresis was carried out withnon-reducing and reducing conditions, respectively. Fab' fragments werefreshly prepared prior to linking to the amphiphilic monolayer.

Fab2 were radiolabelled with 125I to a specific activity of 108 cpm/mgby chloramine T method. 125I Fab2 were incorporated into the unlabelledFab2 to a specific activity of 1×104 cpm per mg unlabelled Fab2 and Fabfragments prepared as described above.

Covalent Attachment of Fab to Lipid and Binding Assay

Pesin digestion of antibody and subsequent reduction of the resultantFab2 to Fab' fragments produces a single reactive thiol group at thecarboxyl terminus of the Fab'. Coupling of this thiol group to the lipidmolecule is achieved via the reaction with a terminal chlorine onpolyethylene oxide attached to the polymerisable lipid molecule.

The monolayer of derivatised lipid was formed by spreading lipid indecane solution on an air-water interface in a Langmuir-Blodgett trough.The nylon peg substrate, previously treated to render surfacehydrophobic, was dipped through the interface so that the hydrocarbonchains of the lipid interacted with the surface of the substrate.

The surface of the trough was cleaned of lipid before the substrate wasquickly withdrawn and transferred to the Fab' solution.

The lipid-coated substrate was immersed into an aqueous solution Fab' ata concentration of 0.1 to 1.0 mg/ml of phosphate buffered saline buffer,pH 8. The reaction between the specific thiol on the Fab' and thechlorine of the lipid polyethylene oxide linker group was carried outfor 3-20 hours at room temperature under N2. 125I Fab' was used as amarker of the reaction as it was carried out on the lipid coatedsubstrate.

The Fab' linked lipid coated substrate was then transferred to amicrotitre well containing 125I-hCG at a concentration of 1 to 5 mg/ml.ph 7.4. The radioactivity of the entire substrate was measured after afifteen minute incubation. Comparison with a conventional immunoassayusing the same amount of antibody in microtitre wells showed that theuse of lipid-Fab coating yielded at least a 2-fold improvement insensitivity.

The same treatment was applied to a palladium-coated glass slidesubstrate, which showed at least a 3-fold increase in sensitivitycompared to conventional immunoassay techniques. A coating of at least1011 Fab molecules pwer cm2 was achieved after incubation times longerthan 10 hours as calculated from radiactivity measurements of 125 I-Fab.

Use of 2 types monoclonal Fab fragments, which bind to two differentsides on the human chorionic gonadotrophin (LCG), gave at least a 50%increase in sensitivity compared to using only one Fab.

EXAMPLE 6 Synthesis of Gramicidin Dimer

A dimer of covalently linked head to head GA molecules having thesequence:

HC-Trp-D-Leu-Trp-D-Leu-Trp-D-Leu-Trp-D-Val-Val-D-Val-Ala-D-Leu-Ala-Gly-Val-Gly-Ala-1-13C-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-d-Leu-Trp-NHCH₂-CH₂ -OH has been synthesised.

Chemicals

Side chain protected BOC-Trp(CHU) and all other BOC amino acids werepurchased from Peptide Institute Inc. (Japan).

1-13C-DL Leucine (1-13C, 99%) was obtained from Cambridge IsotopesLaboratories (Woburn, Mass.). tBOC-Trp(CHO)OCH2PAM resin (0.69 mmol/g)was obtained from Applied Biosystems.

Synthesis

BOC-1-13-D-Leucine was synthesised, according to the procedure of Prasadet al. (Int. J Peptide Protein Res 19, 1982 162-171) with minorvariations from the starting material of 1-13C-DL Leucine.

The 1 13C-D-Leu₁₈ dimer was synthesised by the solid phase method, usinga 430A peptide synthesiser (Applied Biosystems) for the addition of allamino acids except the 1 13 labelled D-Leu which was added manually.

The synthesis started with BOC-Trp(CHO)-OCH2-PAM resin (0.72 g)containing 0.5 mmol of BOC=Trp(CHO) esterified to 1% cross linkedpolystyrene.

The first 6 cycles were single couplings of BOC amino acid will allremaining cycles being doubly coupled.

First couplings were in DMF and recouplings were done with DCM assolvent.

Each amino acid was added with the following steps:

1. Resin washings to DCM.

2. Removal of the BOC group using 33% TFA in DCM for 80 sec., followedby 50% TFA/DCM for 18.5 minutes.

3. 3 DCM washes.

4. Neutralisation with 10% diisopropylethylamine (DIRA) in DMF for 2×1min.

5. 5 DMF washes.

6. 26 min. coupling cycle in DMF via amino acid anhydride (2 fold excessof anhydride) using 2 mmol BOC amino acid and dicyclohexylcarbodiimide(DCC).

7. 5 DCM washes.

Recouple Cycle

1. Wash in coupling solvent (DCM).

2. 10% DIEA in DCM for 30 sec.

3. 5 DCM washes.

4. Recoupling in DCM 30 minutes.

5. 1 DMF wash.

6. 5 DCM washes.

The 1-¹³ C-labelled D-Leu was added to the peptide manually. The resinwas removed from the synthesiser reaction vessel after step 5(neutralisation and washings) of this cycle.

One equivalent (0.5 mmol) of BOC 1-¹³ c-d-Leu was added in 2 ml DCM andstirred for 10 min. One equivalent of DCC in 2 ml of DMF was then addedand allowed to react at room temperature overnight.

The resin was then returned to the synthesiser where it was washed andthen recoupled with unlabelled BOC-D-Leu using the above recouplingcycle.

Resin samples were taken on completion of each cycle in the synthesis todetermine the extent of coupling using quantitative ninhydrin assay(Sarin et al. Analytical Biochemistry, 117, 147-157, 1981). Eachreaction was 99% complete.

The completed peptide was removed from the resin by reaction withethanolamine to give the terminal ethanolamide moiety, followed byde-BOCing and formulation reactions as described by Prasad et al.(1982).

Initial purification of the crude peptide was obtained by filtration inmethanol on a 100 cv×3.2 cm id column of Sephadex LH20 (Pharmacia).

Fractions collected from this column were analysed by reversed phaseHPLC on a radial compression column (8 mm id×10 cm) using either anisocratic aq MeOH solvent (92% MeOH) or a 92% aq MeOH to 100% MeOHgradient.

Analytical TLC's were done on silica gel plates (Merck Kieselgel 60F-254 using solvents.

Chloroform/MeOH/glacial acetic acid 90:10:3 and CHcl₃ /MeOH/H₂ O 65:25:4and Bands were visuallised by ultraviolet light.

The following examples relate to a biosensor fabricated from anamphiphile-ion channel surface attached to a metal electrode. Receptormolecules are covalently linked to the amphiphile-ion channel coating.The binding of the ligand to the receptor molecules act as the gatingmechanism, changing the conductance of the ion channel. The gatingmechanism is related to ion channel concentration and receptorconcentration, as exemplified by the following:

EXAMPLE 7 Synthesis of a Biosensor

A lipid gramicidin surface was prepared on a palladium-coated glasselectrode as described in Example 5. The first monolayer consisted ofdodecane-thiol:gramicidin (ratio 30 to 1) and the second monolayerconsisted of acetate lipid:gramicidin R (at a ratio of 100 to 1). Theformation of the gramicidin R was as described in Example 4.

The electrode was then incubated in an Fab solution consisting of Fabprepared from two monoclonal antibodies to hCG which bind to twodistinct sites on the hCG molecule. The ratio of the two types of Fabwas 1:1. Total concentration of Fab was 0.1 to 1.0 mg/ml of phosphatebuffered saline, pH 8. The electrode was incubated at room temperatuefor 3 to 19 hours. The electrical impedance of the electrode wasmeasured through a frequency range of 1 millihertz to 5 kilohertz, usinga three electrode system, a "Solartron 1250 FRA" impedance analyser andan electro-chemical interface amplifier. Impedance of the lipidgramicidin bilayer was 10⁴.95 ohms at 10 millihertz corresponding to1.6×10⁶ conduting gramicidin channels. (All estimates of number ofconducting channel are based on the gramicidin resistance in black lipidmembranes of 10¹¹ ohms/channel.)

Optimal incubation time was twelve hours in the Fab solution, which gavean increased impedance measurement of 10⁶.15 ohms at 10 millihertzarising from 5.9×10⁴ conducting gramicidin channels (measured at 1millihertz). Washing the electrode in running water and leaving indistilled water for 48 hours did not change the impedance of theelectrode.

The electrode was incubated with hCG in 0.1M NaCl for 15 minutes at 37°C. After washing with distilled water, the electrode was returned to the0.1M NaCl cell and its impedance was measured. An incubation time of 12hours in an Fab solution was found to give the most sensitive change inimpedance upon hCG binding. 0.96 nanograms hCG per ml gave an increasedimpedance of 10⁶.20 ohms at 10 millihertz corresponding to 4.8×10⁴conducting gramicidin channels, measured at 1 millihertz.

Washing the electrode with distilled water or ethanol, did not changethe impedance. Soaking the electrode in distilled water or 0.1M NaCl for24 hours also did not change the impedance of the electrode.

EXAMPLE 8

Palladium-Coating Glass Electrodes were coated using the methoddescribed in Example 7. The first monolayer is as described in Example7, and the second monolayer consisted of total lipid:gramicidin at aratio of 100:1, where the total lipid consisted of acetate lipid:linkerlipid (see Examples 1 to 3) at a ratio of 100:1.

The impdeance of the electrode was measured as described in Example 7.The electrode was incubated in Fab solution for 5 to 19 hours asdescribed in Example 7. A lipid-Fab electrode measured after 5.5 hoursincubation in the Fab solution gave an impedance of 10⁵.4 ohms at 10millihertz corresponding to 1.9×10⁵ conducting gramicidin channels,compared to a lipid-gramicidin only bilayer impedance of 10⁴.6 ohms at10 millihertz.

hCG was incubated with the Fab covered lipid-gramicidin coated electrodeas described in Example 7. The incubation time of 5.5 hours in the Fabsolution was found to give the most sensitive change in impedance uponhCG binding. An impedance of 10⁵.55 ohms at 10 millihertz correspondingto 1.2×10⁵ conducting gramicidin channels was measured after addition of0.96 nanograms hCG per ml. A further addition of hCG to a totalconcentration of 2.56 nanograms per ml increased the impedance in theelectrode to 10⁵.93 ohms at 10 millihertz corresponding to 5.6×10⁵conducting gramicidin channels.

Another electrode with the same coating as described above, gave animpedance measurement of 10⁵.8 ohms at 10 millihertz with 5.5 hours Fabincubation and an impedance measurement of 10⁶.15 ohms at 10 millihertzwith addition of 0.96 nanograms hCG per ml. As a control, addition ofthe same amount of bovine eserum albumin instead of hCG (ie. 1.92×10⁻¹⁴mol per ml) gave an impedance measurement of 10⁵.80 ohms at 10millihertz, equivalent to the lipid-Fab coated electrode without hCG.

EXAMPLE 9

Palladium-coated glass electrodes were coated using the method describedin Example 7. The first monolayer was dodecanethiol:gramicidin A (10:1)and the second monolayer consisted of acetate lipid:gramicidin linker(2:1). The impedance measurements were carried out as described inExample 6.

The bilayer impedance was 10⁵.85 ohms at 1 mHz. Incubation with Fabsolution for 16 hours as described in Example 7 gave an impedance of10⁶.9 ohms at 1 mHz. Addition of 0.08 nanograms hCG/ml gave an impedancemeasurement of 10⁶.56 ohms at 1 mHz.

EXAMPLE 10

Ca²⁺ ions specifically block gramicidan channels. Ca²⁺ ions were addedto the lipid-gramicidin linker coated electrode to test the ability ofCa²⁺ to block gramicidin.

A palladium-coated glass electrode was prepared by the method describedin Example 6 with the first monolayer consisting ofdodecanethiol:gramicidin A (10:1) and second monolayer ofdimyristoylphosphatidylethanolamine:gramicidin A. (1:1). Impedancemeasurements were made as described in Example 7. The bilayer measuredan impedance of 10⁵.05 ohms at 10 mHz. Addition of 50 mM Ca²⁺ ions tothe measuring cell increased the impedance to 10⁵.35 ohms at 10 mHzindicating a decrease in the number of conducting gramicidin channels.

We claim:
 1. A membrane comprising a closely packed array ofself-assembling amphiphilic molecules, the membrane being characterizedin the (1) the membrane includes a plurality of ion channels selectedfrom the group consisting of podands, cryptands and coronands: (2) atleast a proportion of the self assembling amphiphilic molecules comprisea receptor molecule conjugated with a supporting entity, the supportingentity being conjugated with the receptor molecule at a location fromthe receptor site.
 2. A membrane as claimed in claim 1 in which areceptor moiety is attached or bound to the ion channel at an endthereof, the receptor moiety being such that it normally exists in afirst state, but when bound to an analyte exists in a second state, saidchange of state of the receptor moiety causing a change in the abilityof ions to pass through the ion channel.
 3. A membrane as claimed inclaim 2 in which the receptor moiety is an antibody or antibody bindingfragments.
 4. A membrane as claimed in claim 1 in which the receptormolecule is an antibody or antibody binding fragments.
 5. A membrane asclaimed in claim 4 in which the antibody binding fragment is attached toa hydrocarbon chain(s) and in which up to 2% of the amphiphilicmolecules in the membrane comprise an Fab fragment attached to ahydrocarbon chain(s).
 6. A membrane as claimed in claim 1 in which aproportion of amphiphilic molecules comprise a receptor moleculeconjugated with a supporting entity, individual receptor molecules beingreactive with different molecules or antigenic determinants.
 7. Amembrane as claimed in claim 1 in which an antibody or antibody fragmentis attached to the ion channel such that when an antigen is bound to theantibody or antibody fragment ions can pass through the ion channel orare prevented from passing through the channel.
 8. A membrane as claimedin claim 1 in which the amphiphilic molecules, not including a receptormolecule, include or are decorated with at least one moiety cross-linkedwith at least one corresponding moiety on another of these molecules. 9.A membrane as claimed in claim 1 in which the ion channels and/or theproportion of amphiphilic molecules comprising the receptor moleculeconjugated with the supporting entity, each include or are decoratedwith at least one moiety cross-linked with at least one correspondingmoiety on another molecule.
 10. A membrane as claimed in claim 1 inwhich the membrane is attached to a solid surface, the attachment beingby means of groups provided on the membrane, said groups reactive withthe solid surface or groups provided thereon.
 11. A membrane as claimedin claim 2 in which the receptor moiety is a plugging compound whichplugs the ion channel or in which the plugging compound which plugs theion channel is attached to the receptor moiety, the bind of the analyteto the receptor moiety causing a change in the relationship between theplugging compound and the ion channel and altering the ability of ionsto pass through the ion channel.
 12. A membrane as claimed in claim 11in which the plugging compound is a positively charged species with anionic diameter of 4 to 6 Angstroms.
 13. A membrane comprising a closelypacked array of self-assembling amphiphilic molecules, the membraneincluding a plurality of ion channels selected from the group consistingof podands, cryptands and coronands, a receptor moiety being attached tothe ion channel at an end thereof, the receptor moiety being such thatit normally exists in a first state, but when bound to an analyte existsin a second state, said change of state of the receptor moiety causing achange in the ability of ions to pass through the ion channel.
 14. Amembrane as claimed in claim 13 in which the receptor moiety is anantibody or antibody binding fragments.
 15. A membrane as claimed inclaim 13 in which the amphiphilic molecules and/or the ion channels eachinclude or are decorated with at least one moiety cross-linked with atleast one corresponding moiety on another molecule.
 16. A membrane asclaimed in claim 13 in which the membrane is attached to a solidsurface, the attachment being by means of groups provided on themembrane, said groups being reactive with the solid surface or groupsprovided thereon.
 17. A membrane as claimed in claim 13 in which thereceptor moiety is a plugging compound which plugs the ion channel or inwhich a plugging compound which plugs the ion channel is attached to thereceptor moiety, the binding of the analyte to the receptor moietycausing a change in the relationship between the plugging compound andthe ion channel and causing a change in the ability of ions to passthrough the ion channel.
 18. A membrane as claimed in claim 17 in whichthe plugging compound is apositively charged species with an ionicdiameter of 4 to 6 Angstroms.
 19. A membrane comprising a closely packedarray of self-assembling amphiphilic molecules, the membrane beingcharacterized in that (1) the membrane includes a plurality of ionchannels selected from the group consisting of peptides capable offorming helices and aggregates thereof; and (2) at least a proportion ofthe self assembling amphiphilic molecules comprise a receptor moleculeconjugated with a supporting entity, the receptor molecule having areceptor site, the supporting entity being selected from the groupconsisting of hydrocarbon chains having lipid head groups andcross-linkable molecules, the supporting entity being conjugated withthe receptor molecule at a location remote from the receptor site.
 20. Amembrane as claimed in claim 19 in which the ion channels are aggregatesof α helical peptides.
 21. A membrane as claimed in claim 19 in whichthe ion channels are peptides which form a β helix.
 22. A membrane asclaimed in claim 21 in which the ion channels are gramicidin oranalogues thereof.
 23. A membrane as claimed in claim 21 in which theion channel is gramicidin A or analogues thereof.
 24. A membrane asclaimed in claim 19 in which a receptor moiety is attached or bound withthe ion channel at an end thereof, the receptor moiety being such thatit normally exists in a first state, but when bound to an analyte existsin a second state, said change of state of the receptor moiety causing achange in the ability of ions to pass through the ion channel.
 25. Amembrane as claimed in claim 24 in which the receptor moiety is anantibody or antibody binding fragments.
 26. A membrane as claimed inclaim 19 in which the receptor molecule is an antibody or antibodybinding fragments.
 27. A membrane as claimed in claim 19 in which aproportion of amphiphilic molecules comprise a receptor moleculeconjugated with a supporting entity, individual receptor molecules beingreactive with different molecules or antigenic determinants.
 28. Amembrane as claimed in claim 19 in which an antibody or antibodyfragment is attached to the ion channel such that when an antigen isbound to the antibody or antibody binding fragments the ability of ionsto pass through the ion channel is altered.
 29. A membrane as claimed inclaim 19 in which the amphiphilic molecules, not including a receptormolecule, include or are decorated with at least one moiety cross-linkedwith at least one corresponding moiety on another of these molecules.30. A membrane as claimed in claim 19 in which the ion channels and/orthe proportion of amphiphilic molecules comprising the receptor moleculeconjugated with the supporting entity, each include or are decoratedwith at least one moiety cross-linked with at least one correspondingmoiety on another molecule.
 31. A membrane as claimed in claim 19 inwhich the membrane is attached to a solid surface, the attachment beingby means of groups provded on the membrane, said groups reactive withthe solid surface or groups provided thereon.
 32. A membrane as claimedin claim 24 in which the receptor moiety is a plugging compound whichplugs the ion channel or in which a plugging compound which plugs theion channel is attached to the receptor moiety, the binding of theanalyte to the receptor moiety causing a change in the relationshipbetween the plugging compound and the ion channel and causing a changein the ability of ions to pass through the ion channel.
 33. A membraneas claimed in claim 32 in which the plugging compound is a positivelycharged species with an ionic diameter of 4 to 6 Angstroms.
 34. Amembrane as claimed in claim 24 in which the ion channel is dimericgramicidin A and the receptor moiety is an antibody fragment, theability of ions to pass through the ion channel being altered upon thebinding of the antibody fragment to an analyte due to the disruption ofthe dimeric gramicidin A backbone, or to the disruption of the portionof the helix of the dimeric gramicidin attached to an Fab fragment. 35.A membrane as claimed in claim 2 in which the membrane is a bilayer. 36.A membrane as claimed in claim 13 in which the membrane is a bilayer.37. A membrane as claimed in claim 19 in which the membrane is abilayer.
 38. A membrane as claimed in claim 2 in which the membrane is amonolayer.
 39. A membrane as claimed in claim 13 in which the membraneis a monolayer.
 40. A membrane as claimed in claim 19 in which themembrane is a monolayer.
 41. A membrane as claimed in claim 2 in which aproportion of the amphiphilic molecules are membrane spanning lipids.42. A membrane as claimed in claim 13 in which a proportion of theamphiphilic molecules are membrane spanning lipids.
 43. A membrane asclaimed in claim 19 in which a proportion of the amphiphilic moleculesare membrane spanning lipids.
 44. A membrane as claimed in claim 2 inwhich the receptor moiety is attached to the ion channel via anon-hydrophobic linker group.
 45. A membrane as claimed in claim 13 inwhich the receptor moiety is attached to the ion channel via anon-hydrophobic linker group.
 46. A membrane as claimed in claim 24 inwhich the receptor moiety is attached to the ion channel via anon-hydrophobic linker group.
 47. A membrane as claimed in claim 2 inwhich the receptor moiety is attached to the supporting entity via anon-hydrophobic linker group.
 48. A membrane as claimed in claim 19 inwhich the receptor moiety is attached to the supporting entity via anon-hydrophobic linker group.
 49. A membrane as claimed in claim 2 inwhich the receptor moiety is attached to the supporting entity via anon-hydrophobic linker group.
 50. A membrane as claimed in claim 24 inwhich the receptor moiety binds to the receptor molecule.
 51. Themembrane of claim 1, wherein the receptor molecule is selected from thegroup consisting of antigens, immunoglobulins, peptides, antibodies,antibody binding fragments, enzymes, dyes, and lectins.
 52. The membraneof claim 13, wherein the receptor moiety is selected from the groupconsisting of antigens, immunoglobulins, peptides, antibodies, antibodybinding fragments, enzymes, dyes, and lectins.
 53. The membrane of claim19, wherein the receptor molecule is selected from the group consistingof antigens, immunoglobulines, peptides, antibodies, antibody bindingfragments, enzymes, dyes, and lectins.